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1.
The role of ATP and its stable analogue ATPγS [adenosine-5′-o-(3-thio)triphosphate] was studied in rat hippocampal neurotransmission
under normoxic conditions and during oxygen and glucose deprivation (OGD). Field excitatory postsynaptic potentials (fEPSPs)
from the dendritic layer or population spikes (PSs) from the soma were extracellularly recorded in the CA1 area of the rat
hippocampus. Exogenous application of ATP or ATPγS reduced fEPSP and PS amplitudes. In both cases the inhibitory effect was
blocked by the selective A 1 adenosine receptor antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine) and was potentiated by different ecto-ATPase inhibitors:
ARL 67156 (6- N, N-diethyl- D-β,γ-dibromomethylene), BGO 136 (1-hydroxynaphthalene-3,6-disulfonate) and PV4 [hexapotassium dihydrogen monotitanoundecatungstocobaltate(II)
tridecahydrate, K 6H 2[TiW 11CoO 40]·13H 2O]. ATPγS-mediated inhibition was reduced by the P2 antagonist suramin [8-(3-benzamido-4-methylbenzamido)naphthalene-1,3,5-trisulfonate]
at the somatic level and by other P2 blockers, PPADS (pyridoxalphosphate-6-azophenyl-2′,4′-disulfonate) and MRS 2179 (2′-deoxy- N
6-methyladenosine 3′,5′-bisphosphate), at the dendritic level. After removal of both P2 agonists, a persistent increase in
evoked synaptic responses was recorded both at the dendritic and somatic levels. This effect was prevented in the presence
of different P2 antagonists. A 7-min OGD induced tissue anoxic depolarization and was invariably followed by irreversible
loss of fEPSP. PPADS, suramin, MRS2179 or BBG (brilliant blue G) significantly prevented the irreversible failure of neurotransmission
induced by 7-min OGD. Furthermore, in the presence of these P2 antagonists, the development of anoxic depolarization was blocked
or significantly delayed. Our results indicate that P2 receptors modulate CA1 synaptic transmission under normoxic conditions
by eliciting both inhibitory and excitatory effects. In the same brain region, P2 receptor stimulation plays a deleterious
role during a severe OGD insult. 相似文献
2.
Extracellular nucleotides and their metabolites activate ionotropic P2X and metabotropic P2Y receptors on the surface of various types of cells. Here, we investigated the involvement of P2X and P2Y receptor-mediated signaling in TCR-dependent T cell activation. Murine T cells were activated by stimulation of TCR, and both CD25 expression and interleukin (IL)-2 production were observed in activated T cells. Ecto-nucleotidase apyrase and P2Y 6 antagonist MRS2578 significantly blocked the increases of both CD25 expression and IL-2 production, and P2X 7 antagonists A438079 and oxidized ATP inhibited IL-2 production rather than CD25 expression, suggesting the involvement of P2Y 6 and P2X 7 receptors in different processes of T cell activation. MRS2578 also blocked TCR-dependent elevation of cytosolic Ca 2+ in T cells. The P2X 7 and P2Y 6 receptors were expressed in murine CD4 T cells. In conclusion, our results indicate that activation of P2Y 6 and P2X 7 receptors contributes to T cell activation via TCR. 相似文献
3.
Extracellular ATP triggers changes in intracellular Ca 2+, ion channel function, and membrane trafficking in adipocytes. The aim of the present study was to determine which P2 receptors
might mediate the Ca 2+ signaling and membrane trafficking responses to ATP in brown fat cells. RT-PCR was used to determine which P2 receptors are
expressed in brown fat cells. Responses to nucleotide agonists and antagonists were characterized using fura-2 fluorescence
imaging of Ca 2+ responses, and FM 1-43 fluorescence imaging and membrane capacitance measurements to assess membrane trafficking. The pharmacology
of the Ca 2+ responses fits the properties of the P2Y receptors for which mRNA is expressed, but the agonist and antagonist sensitivity
of the membrane-trafficking response was not consistent with any P2 receptor described to date. Brown adipocytes expressed
mRNA for P2Y 2, P2Y 6, and P2Y 12 metabotropic receptors and P2X 1, P2X 2, P2X 3, P2X 4, P2X 5, and P2X 7 ionotropic receptors. The agonists ATP, ADP, UTP, UDP and 2′, 3′-(benzoylbenzoyl) ATP (BzATP) increased intracellular Ca 2+, while 100 μ M suramin, pyridoxal-phosphate-6-azophenyl-2′ 4′-disulfonic acid (PPADS), or Reactive Blue 2 partially blocked Ca 2+ responses. ATP, but not ADP, UTP, UDP or BzATP activated membrane trafficking. The membrane response could be blocked completely
with 1 μ M PPADS but not by the antagonist MRS2179. We conclude that multiple P2 receptors mediate the ATP responses of brown fat cells,
and that membrane trafficking is regulated by a P2 receptor showing unusual properties. 相似文献
4.
The physiological mechanisms that regulate reactive hyperemia are not fully understood. We postulated that the endothelial P2Y 1 receptor that release vasodilatory factors in response to ADP might play a vital role in the regulation of coronary flow. Intracoronary flow was measured with a Doppler flow-wire in a porcine model. 2-MeSADP (10 –5 M), ATP (10 –4 M) or UTP (10 –4 M) alone or as co-infusion with a selective P2Y 1 receptor blocker, MRS 2179 (10 –3 M) was locally delivered through the tip of a coronary angioplasty balloon. In separate pigs the coronary artery was occluded with the balloon for 10 min. During the first and tenth minutes of coronary ischemia, 2.5 ml of MRS 2179 (10 –3 M) was delivered distal to the occlusion in 8 pigs, 10 pigs were used as controls. MRS 2179 fully inhibited the 2-MeSADP-mediated coronary flow increase ( P < 0.05) with no effect on UTP, indicating selective P2Y 1 inhibition. ATP-mediated flow increase was significantly inhibited by MRS 2179. During reactive hyperemia following coronary occlusion, flow increased by nearly sevenfold. MRS 2179, however, reduced the post-ischemic hyperemia by a mean of 46% during the period 1–2.5 min following balloon deflation ( P < 0.05), which corresponds to peak velocity flow during reperfusion. In conclusion, MRS 2179, a selective P2Y 1 receptor blocker, significantly reduces the increased coronary flow caused both by 2-MeSADP and reactive hyperemia in coronary arteries. Thus, ADP acting on the endothelial P2Y 1 receptor may play a major role in coronary flow during post-ischemic hyperemia. 相似文献
5.
Tanshinone I (TsI), a lipophilic diterpene extracted from Danshan (Radix Salvia miltiorrhizae), exerts neuroprotection in cerebrovascular diseases including transient ischemic attack. In this study, we examined effects of TsI on cell proliferation and neuronal differentiation in the subgranular zone (SGZ) of the mouse dentate gyrus (DG) using Ki-67, BrdU and doublecortin (DCX) immunohistochemistry. Mice were treated with 1 and 2 mg/kg TsI for 28 days. In the 1 mg/kg TsI-treated-group, distribution patterns of BrdU, Ki-67 and DCX positive ( +) cells in the SGZ were similar to those in the vehicle-treated-group. However, in the 2 mg/kg TsI-treated-group, double labeled BrdU +/NeuN + cells, which are mature neurons, as well as Ki-67 +, DCX + and BrdU + cells were significantly increased compared with those in the vehicle-treated-group. On the other hand, immunoreactivities and protein levels of Wnt-3, β-catenin and serine-9-glycogen synthase kinase-3β (p-GSK-3β), which are related with morphogenesis, were significantly increased in the granule cell layer of the DG only in the 2 mg/kg TsI-treated-group. Therefore, these findings indicate that TsI can promote neurogenesis in the mouse DG and that the neurogenesis is related with increases of Wnt-3, p-GSK-3β and β-catenin immunoreactivities. 相似文献
6.
Neuro-2a (N2a) cells are derived from spontaneous neuroblastoma of mouse and capable to differentiate into neuronal-like cells. Recently, P2X 7 receptor has been shown to sustain growth of human neuroblastoma cells but its role during neuronal differentiation remains unexamined. We characterized the role of P2X 7 receptors in the retinoic acid (RA)-differentiated N2a cells. RA induced N2a cells differentiation into neurite bearing and neuronal specific proteins, microtubule-associated protein 2 (MAP2) and neuronal specific nuclear protein (NeuN), expressing neuronal-like cells. Interestingly, the RA-induced neuronal differentiation was associated with decreases in the expression and function of P2X 7 receptors. Functional inhibition of P2X 7 receptors by P2X 7 receptor selective antagonists, 5′-triphosphate, periodate-oxidized 2′,3′-dialdehyde ATP (oATP), brilliant blue G (BBG) or A438079 induced neurite outgrowth. In addition, RA and oATP treatment stimulated the expression of neuron-specific class III beta-tubulin (TuJ1), and knockdown of P2X 7 receptor expression by siRNA induced neurite outgrowth. To elucidate the possible mechanism, we found the levels of basal intracellular Ca 2+ concentrations ([Ca 2+] i) were decreased in either RA- or oATP-differentiated or P2X 7 receptor knockdown N2a cells. Simply cultured N2a cells in low Ca 2+ medium induced a 2-fold increase in neurite length. Treatment of N2a cells with ATP hydrolase apyrase and the P2X 7 receptors selective antagonist oATP or BBG decreased cell viability and cell number. Nevertheless, oATP but not BBG decreased cell proliferation and cell cycle progression. These results suggest for the first time that decreases in expression/function of P2X 7 receptors are involved in neuronal differentiation. We provide additional evidence shown that the ATP release-activated P2X 7 receptor is important in maintaining cell survival of N2a neuroblastoma cells. 相似文献
7.
P2Y receptors have been implicated in the calcium mobilization by the response to neuroexcitatory substances in neurons and astrocytes, but little is known about P2Y receptors in microglia cells. In the present study, the effects of ADP on the intracellular calcium concentration ([Ca 2+]i) in cultured dorsal spinal cord microglia were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescence indicator that could monitor real-time alterations of [Ca 2+]i. Here we show that ADP (0.01–100 μM) causes a rapid increase in [Ca 2+]i with a dose-dependent manner in cultured microglia. The action of ADP on [Ca 2+]i was significantly blocked by MRS2211 (a selective P2Y 13 receptor antagonist), but was unaffected by MRS2179 (a selective P2Y 1 receptor antagonist) or MRS2395 (a selective P2Y 12 receptor antagonist), which suggest that P2Y 13 receptor may be responsible for ADP-evoked Ca 2+ mobilization in cultured microglia. P2Y 13-evoked Ca 2+ response can be obviously inhibited by BAPTA-AM and U-73122, respectively. Moreover, removal of extracellular Ca 2+ (by EGTA) also can obvious suppress the Ca 2+ mobilization. These results means both intracellular calcium and extracellular calcium are potentially important mechanisms in P2Y 13 receptor-evoked Ca 2+ mobilization. However, P2Y 13 receptor-evoked Ca 2+ response was not impaired after CdCl 2 and verapamil administration, which suggest that voltage-operated Ca 2+ channels may be not related with P2Y 13-evoked Ca 2+ response. In addition, Ca 2+ mobilization induced by ADP was abolished by different store-operated Ca 2+ channels (SOCs) blocker, 2-APB (50 μM) and SKF-96365 (1 mM), respectively. These observations suggest that the activation of P2Y 13 receptor might be involved in the effect of ADP on [Ca 2+]i in cultured dorsal spinal cord microglia. Furthermore, our results raise a possibility that P2Y 13 receptor activation causes Ca 2+ release from Ca 2+ store, which leads to the opening of SOCs. 相似文献
8.
During exercise, skeletal muscle produces reactive oxygen species (ROS) via NADPH oxidase (NOX2) while inducing cellular adaptations associated with contractile activity. The signals involved in this mechanism are still a matter of study. ATP is released from skeletal muscle during electrical stimulation and can autocrinely signal through purinergic receptors; we searched for an influence of this signal in ROS production. The aim of this work was to characterize ROS production induced by electrical stimulation and extracellular ATP. ROS production was measured using two alternative probes; chloromethyl-2,7- dichlorodihydrofluorescein diacetate or electroporation to express the hydrogen peroxide-sensitive protein Hyper. Electrical stimulation (ES) triggered a transient ROS increase in muscle fibers which was mimicked by extracellular ATP and was prevented by both carbenoxolone and suramin; antagonists of pannexin channel and purinergic receptors respectively. In addition, transient ROS increase was prevented by apyrase, an ecto-nucleotidase. MRS2365, a P2Y 1 receptor agonist, induced a large signal while UTPyS (P2Y 2 agonist) elicited a much smaller signal, similar to the one seen when using ATP plus MRS2179, an antagonist of P2Y 1. Protein kinase C (PKC) inhibitors also blocked ES-induced ROS production. Our results indicate that physiological levels of electrical stimulation induce ROS production in skeletal muscle cells through release of extracellular ATP and activation of P2Y1 receptors. Use of selective NOX2 and PKC inhibitors suggests that ROS production induced by ES or extracellular ATP is mediated by NOX2 activated by PKC. 相似文献
9.
Glucose uptake by peripheral tissues such as skeletal muscles and adipocytes is important in the maintenance of glucose homeostasis. We previously demonstrated that P2Y 6 receptor (P2Y 6R) agonists protect pancreatic islet cells from apoptosis and stimulate glucose-dependent insulin release. Here, we investigated the effects of P2Y 6R activation on glucose uptake in insulin target tissues. An agonist of the P2Y 6R, P 1-(5′-uridine)-P 3-(5′- N4-methoxycytidine)-triphosphate (MRS2957), significantly increased the uptake of [ 3H]2-deoxyglucose in mouse C2C12 myotubes and 3T3-L1 adipocytes, and this stimulation was significantly decreased by a selective P2Y 6R antagonist N, N″-1,4-butanediyl- bis[ N′-(3-isothiocyanatophenyl)thiourea] (MRS2578). Pre-incubation with Compound C (an inhibitor of 5′-AMP-activated protein kinase, AMPK), or AMPK siRNA abolished the stimulatory effect of MRS2957 on glucose uptake. Also, MRS2957 (60 min incubation) increased recruitment of the facilitated glucose transporter-4 (GLUT4) to the cell membrane, which was blocked by MRS2578. Treatment of C2C12 myotubes with MRS2957 induced significant phosphorylation of AMPK, which increase GLUT4 expression through histone deacetylase (HDAC)5 signaling. Glucose uptake in primary mouse adipocytes from wild-type mice was stimulated upon P2Y 6R activation by either MRS2957 or native agonist UDP, and the P2Y 6R effect was antagonized by MRS2578. However, in adipocytes from P2Y 6R-knockout mice P2Y 6R agonists had no effect on glucose uptake, and there was no change in the glucose uptake by insulin. Our results indicate that the P2Y 6R promotes glucose metabolism in peripheral tissues, which may be mediated through AMPK signaling. 相似文献
10.
Splanchnic angiogenesis in liver cirrhosis often leads to complications as gastroesophageal variceal hemorrhage and the treatment efficacy is adversely affected by poor portal-systemic collateral vasoresponsiveness related to nitric oxide (NO). Purinergic receptor subtype P2X 7 participates in the modulation of inflammation, angiogenesis, fibrogenesis and vasoresponsiveness, but the relevant influence in cirrhosis is unknown. Common bile duct-ligated (CBDL) or sham-operated Spraque-Dawley rats received brilliant blue G (BBG, a P2X 7 antagonist and food additive) or vehicle from the 15 th to 28 th day after operations, then hemodynamics, mesenteric angiogenesis, portal-systemic shunting, liver fibrosis, and protein expressions of angiogenic and fibrogenic factors were evaluated. The influence of oxidized ATP (oATP, another P2X 7 receptor antagonist) on the collateral vasoresponsiveness to arginine vasopressin (AVP) was also surveyed. BBG decreased superior mesenteric artery (SMA) flow, portal-systemic shunting, mesenteric vascular density, and mesenteric protein expressions of vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), phospho (p)-VEGFR2, platelet-derived growth factor (PDGF), PDGF receptor beta (PDGFRβ), cyclooxygenase (COX)-1, COX-2, and endothelial NO synthase (eNOS) in CBDL rats. BBG also ameliorated liver fibrosis and down-regulated hepatic interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), PDGF, IL-1β, transforming growth factor-beta (TGF-β), p-extracellular-signal-regulated kinases (ERK), and alpha-smooth muscle actin (α-SMA) expressions in CBDL rats. The collateral vasocontractility to AVP was enhanced by oATP. oATP down-regulated eNOS, inducible NOS (iNOS), VEGF, Akt, p-Akt, and nuclear factor-kappa B (NF-κB) expressions in splenorenal shunt, the most prominent intra-abdominal collateral vessel in rodents. P2X 7 antagonism alleviates splanchnic hyperemia, severity of portal-systemic shunting, mesenteric angiogenesis, liver fibrosis, and enhances portal-systemic collateral vasoresponsiveness in cirrhotic rats. P2X 7 blockade may be a feasible strategy to control cirrhosis and complications. 相似文献
11.
Adenosine triphosphate (ATP) participates in signal transmission by acting on P2X receptors, and the P2X 7 receptor is involved in the pathophysiological changes of ischemic injury. The PC12 cell line is a popular model system to study sympathetic neuronal function. Long noncoding RNAs (lncRNAs) are highly expressed in the nervous system and serve as regulatory RNAs. In this study, the effects of NONRATT021972 lncRNA siRNA on P2X 7-mediated PC12 neuronal injury after exposure to oxygen-glucose deprivation (OGD) were investigated. Our results showed that the viability of PC12 cells cultured with OGD or the P2X 7 agonist BzATP was significantly decreased. Treatment with NONRATT021972 siRNA reversed the decreased viability of PC12 cells under OGD conditions. The upregulated P2X 7 mRNA and protein levels in PC12 cells under OGD conditions or BzATP treatment were significantly decreased when pretreated with NONRATT021972 siRNA. Moreover, NONRATT021972 siRNA treatment effectively suppressed the increase in [Ca 2+] i induced by OGD or P2X 7 agonists (ATP or BzATP) in PC12 cells. Therefore, treatment with NONRATT021972 siRNA may decrease sympathetic neuronal injury induced by ischemia. 相似文献
12.
Microglial cells are the primary immune effector cells in the brain. Extracellular ATP, e.g., released after brain injury,
may initiate microglial activation via stimulation of purinergic receptors. In the rat nucleus accumbens (NAc), the involvement
of P2X and P2Y receptors in the generation of microglial reaction in vivo was investigated. A stab wound in the NAc increased
immunoreactivity (IR) for P2X 1,2,4,7 and P2Y 1,2,4,6,12 receptors on microglial cells when visualized with confocal laser scanning microscopy. A prominent immunolabeling of P2X 7 receptors with antibodies directed against the ecto- or endodomain was found on Griffonia simplicifolia isolectin-B4-positive cells. Additionally, the P2X 7 receptor was colocalized with active caspase 3 but not with the anti-apoptotic marker pAkt. Four days after local application
of the agonists α,βmeATP, ADPβS, 2MeSATP, and BzATP, an increase in OX 42- and G. simplicifolia isolectin-IR was observed around the stab wound, quantified both densitometrically and by counting the number of ramified
and activated microglial cells, whereas UTPγS appeared to be ineffective. The P2 receptor antagonists PPADS and BBG decreased
the injury-induced increase of these IRs when given alone and in addition inhibited the agonist effects. Further, the intra-accumbally
applied P2X 7 receptor agonist BzATP induced an increase in the number of caspase-3-positive cells. These results indicate that ATP, acting
via different P2X and P2Y receptors, is a signaling molecule in microglial cell activation after injury in vivo. The up-regulation
of P2X 7-IR after injury suggests that this receptor is involved in apoptotic rather than proliferative effects. 相似文献
13.
Methylmercury (MeHg) is a well known environmental pollutant that induces serious neuronal damage. Although MeHg readily crosses the blood-brain barrier, and should affect both neurons and glial cells, how it affects glia or neuron-to-glia interactions has received only limited attention. Here, we report that MeHg triggers ATP/P2Y 1 receptor signals in astrocytes, thereby protecting neurons against MeHg via interleukin-6 (IL-6)-mediated pathways. MeHg increased several mRNAs in astrocytes, among which IL-6 was the highest. For this, ATP/P2Y 1 receptor-mediated mechanisms were required because the IL-6 production was (i) inhibited by a P2Y 1 receptor antagonist, MRS2179, (ii) abolished in astrocytes obtained from P2Y 1 receptor-knockout mice, and (iii) mimicked by exogenously applied ATP. In addition, (iv) MeHg released ATP by exocytosis from astrocytes. As for the intracellular mechanisms responsible for IL-6 production, p38 MAP kinase was involved. MeHg-treated astrocyte-conditioned medium (ACM) showed neuro-protective effects against MeHg, which was blocked by anti-IL-6 antibody and was mimicked by the application of recombinant IL-6. As for the mechanism of neuro-protection by IL-6, an adenosine A 1 receptor-mediated pathway in neurons seems to be involved. Taken together, when astrocytes sense MeHg, they release ATP that autostimulates P2Y 1 receptors to upregulate IL-6, thereby leading to A 1 receptor-mediated neuro-protection against MeHg. 相似文献
14.
To assess the putative role of adenosine triphosphate (ATP) upon nitric oxide (NO) production in the hippocampus, we used as a model both rat hippocampal slices and isolated hippocampal neurons in culture, lacking glial cells. In hippocampal slices, additions of exogenous ATP or 2′(3′)-O-(4-Benzoylbenzoyl) ATP (Bz-ATP) elicited concentration-dependent NO production, which increased linearly within the first 15 min and plateaued thereafter; agonist EC 50 values were 50 and 15 µM, respectively. The NO increase evoked by ATP was antagonized in a concentration-dependent manner by Coomassie brilliant blue G (BBG) or by N ω-propyl-L-arginine, suggesting the involvement of P2X 7Rs and neuronal NOS, respectively. The ATP induced NO production was independent of N-methyl-D-aspartic acid (NMDA) receptor activity as effects were not alleviated by DL-2-Amino-5-phosphonopentanoic acid (APV), but antagonized by BBG. In sum, exogenous ATP elicited NO production in hippocampal neurons independently of NMDA receptor activity. 相似文献
15.
Various radioligands have been used to characterize and quantify the platelet P2Y 12 receptor, which share several weaknesses: (a) they are metabolically unstable and substrates for ectoenzymes, (b) they are agonists, and (c) they do not discriminate between P2Y 1 and P2Y 12. We used the [ 3H]PSB-0413 selective P2Y 12 receptor antagonist radioligand to reevaluate the number of P2Y 12 receptors in intact platelets and in membrane preparations. Studies in humans showed that: (1) [ 3H]PSB-0413 bound to 425 ± 50 sites/platelet ( KD = 3.3 ± 0.6 nM), (2) 0.5 ± 0.2 pmol [ 3H]PSB-0413 bound to 1 mg protein of platelet membranes ( KD = 6.5 ± 3.6 nM), and (3) competition studies confirmed the known features of P2Y 12, with the expected rank order of potency: AR-C69931MX > 2MeSADP ≫ ADPβS > ADP, while the P2Y 1 ligand MRS2179 and the P2X 1 ligand α,β-Met-ATP did not displace [ 3H]PSB-0413 binding. Patients with severe P2Y 12 deficiency displayed virtually no binding of [ 3H]PSB-0413 to intact platelets, while a patient with a dysfunctional P2Y 12 receptor had normal binding. Studies in mice showed that: (1) [ 3H]PSB-0413 bound to 634 ± 87 sites/platelet ( KD = 14 ± 4.5 nM) and (2) 0.7 pmol ± 0.3 [ 3H]PSB-0413 bound to 1 mg protein of platelet membranes ( KD = 9.1 ± 5.3 nM). Clopidogrel and other thiol reagents like pCMBS or DTT abolished the binding both to intact platelets and membrane preparations. Therefore, [ 3H]PSB-0413 is an accurate and selective tool for radioligand binding studies aimed at quantifying P2Y 12 receptors, to identify patients with P2Y 12 deficiencies or quantify the effect of P2Y 12 targeting drugs. 相似文献
16.
Rapid phosphoester hydrolysis of endogenous purine and pyrimidine nucleotides has challenged the characterization of the role of P2 receptors in physiology and pathology. Nucleotide phosphoester stabilization has been pursued on a number of medicinal chemistry fronts. We investigated the in vitro and in vivo stability and pharmacokinetics of prototypical nucleotide P2Y1 receptor (P2Y1R) agonists and antagonists. These included the riboside nucleotide agonist 2-methylthio-ADP and antagonist MRS2179, as well as agonist MRS2365 and antagonist MRS2500 containing constrained (N)-methanocarba rings, which were previously reported to form nucleotides that are more slowly hydrolyzed at the α-phosphoester compared with the ribosides. In vitro incubations in mouse and human plasma and blood demonstrated the rapid hydrolysis of these compounds to nucleoside metabolites. This metabolism was inhibited by EDTA to chelate divalent cations required by ectonucleotidases for nucleotide hydrolysis. This rapid hydrolysis was confirmed in vivo in mouse pharmacokinetic studies that demonstrate that MRS2365 is a prodrug of the nucleoside metabolite AST-004 (MRS4322). Furthermore, we demonstrate that the nucleoside metabolites of MRS2365 and 2-methylthio-ADP are adenosine receptor (AR) agonists, notably at A3 and A1ARs. In vivo efficacy of MRS2365 in murine models of traumatic brain injury and stroke can be attributed to AR activation by its nucleoside metabolite AST-004, rather than P2Y1R activation. This research suggests the importance of reevaluation of previous in vitro and in vivo research of P2YRs and P2XRs as there is a potential that the pharmacology attributed to nucleotide agonists is due to AR activation by active nucleoside metabolites. 相似文献
17.
Our work aimed to provide a topographical analysis of all known ionotropic P2X 1–7 and metabotropic P2Y 1,2,4,6,11–14 receptors that are present in vivo at the protein level in the basal ganglia nuclei and particularly in rat brain slices
from striatum and substantia nigra. By immunohistochemistry-confocal and Western blotting techniques, we show that, with the
exception of P2Y 11,13 receptors, all other subtypes are specifically expressed in these areas in different amounts, with ratings of low (P2X 5,6 and P2Y 1,6,14 in striatum), medium (P2X 3 in striatum and substantia nigra, P2X 6,7 and P2Y 1 in substantia nigra) and high. Moreover, we describe that P2 receptors are localized on neurons (colocalizing with neurofilament
light, medium and heavy chains) with features that are either dopaminergic (colocalizing with tyrosine hydroxylase) or GABAergic
(colocalizing with parvalbumin and calbindin), and they are also present on astrocytes (P2Y 2,4, colocalizing with glial fibrillary acidic protein). In addition, we aimed to investigate the expression of P2 receptors
after dopamine denervation, obtained by using unilateral injection of 6-hydroxydopamine as an animal model of Parkinson’s
disease. This generates a rearrangement of P2 proteins: most P2X and P2Y receptors are decreased on GABAergic and dopaminergic
neurons, in the lesioned striatum and substantia nigra, respectively, as a consequence of dopaminergic denervation and/or
neuronal degeneration. Conversely, P2X 1,3,4,6 on GABAergic neurons and P2Y 4 on astrocytes augment their expression exclusively in the lesioned substantia nigra reticulata, probably as a compensatory
reaction to dopamine shortage. These results disclose the presence of P2 receptors in the normal and lesioned nigro-striatal
circuit, and suggest their potential participation in the mechanisms of Parkinson’s disease. 相似文献
18.
In the present study, we compared differences in cell proliferation, neuroblast differentiation and neuronal maturation in
the hippocampal dentate gyrus (DG) between the adult and aged gerbil induced by 5 min of transient global cerebral ischemia
using Ki-67 and BrdU (markers for cell proliferation), doublecortin (DCX, a marker for neuroblast differentiation) and neuronal
nuclei (NeuN, a marker for mature neuron). The number of Ki-67-immunoreactive ( +) cells in the DG of both the groups peaked 7 days after ischemia/reperfusion (I/R). However, the number in the aged DG was
40.6 ± 1.8% of that in the adult DG. Thereafter, the number decreased with time. After ischemic damage, DCX immunoreactivity
and its protein level in the adult and aged DG peaked at 10 and 15 days post-ischemia, respectively. However, DCX immunoreactivity
and its protein levels in the aged DG were much lower than those in the adult. DCX immunoreactivity and its protein level
in the aged DG were 11.1 ± 0.6% and 34.4 ± 2.1% of the adult DG, respectively. In addition, the number of Ki-67 + cells and DCX immunoreactivity in both groups were similar to those in the sham at 60 days postischemia. At 30 days post-ischemia,
the number of BrdU + cells and BrdU +/NeuN + cells in the adult-group were much higher (281.2 ± 23.4% and 126.4 ± 7.4%, respectively) than the aged-group (35.6 ± 6.8%
and 79.5 ± 6.1%, respectively). These results suggest that the ability of neurogenesis in the ischemic aged DG is much lower
than that in the ischemic adult DG. 相似文献
19.
Here we elaborated an analytical approach for the simulation of dose-response curves mediated by cellular receptors coupled to PLC and Ca 2+ mobilization. Based on a mathematical model of purinergic Ca 2+ signaling in taste cells, the analysis of taste cells responsiveness to nucleotides was carried out. Consistently with the expression of P2Y 2 and P2Y 4 receptors in taste cells, saturating ATP and UTP equipotently mobilized intracellular Ca 2+. Cellular responses versus concentration of BzATP, a P2Y 2 agonist and a P2Y 4 antagonist, implicated high and low affinity BzATP receptors. Suramin modified the BzATP dose-response curve in a manner that suggested the low affinity receptor to be weakly sensitive to this P2Y antagonist. Given that solely P2Y 2 and P2Y 11 are BzATP receptors, their high sensitivity to suramin is poorly consistent with the suramin effects on BzATP responses. We simulated a variety of dose-response curves for different P2Y receptor sets and found that the appropriate fit of the overall pharmacological data was achievable only with dimeric receptors modeled as P2Y 2/P2Y 4 homo- and heterodimers. Our computations and analytical analysis of experimental dose-response curves raise the possibility that ATP responsiveness of mouse taste cells is mediated by P2Y 2 and P2Y 4 receptors operative mostly in the dimeric form. 相似文献
20.
Endosomal trafficking is intricately linked to G protein-coupled receptors (GPCR) fate and signaling. Extracellular uridine diphosphate (UDP) acts as a signaling molecule by selectively activating the GPCR P2Y 6. Despite the recent interest for this receptor in pathologies, such as gastrointestinal and neurological diseases, there is sparse information on the endosomal trafficking of P2Y 6 receptors in response to its endogenous agonist UDP and synthetic selective agonist 5-iodo-UDP (MRS2693). Confocal microscopy and cell surface ELISA revealed delayed internalization kinetics in response to MRS2693 vs. UDP stimulation in AD293 and HCT116 cells expressing human P2Y 6. Interestingly, UDP induced clathrin-dependent P2Y 6 internalization, whereas receptor stimulation by MRS2693 endocytosis appeared to be associated with a caveolin-dependent mechanism. Internalized P2Y 6 was associated with Rab4, 5, and 7 positive vesicles independent of the agonist. We have measured a higher frequency of receptor expression co-occurrence with Rab11-vesicles, the trans-Golgi network, and lysosomes in response to MRS2693. Interestingly, a higher agonist concentration reversed the delayed P2Y 6 internalization and recycling kinetics in the presence of MRS2693 stimulation without changing its caveolin-dependent internalization. This work showed a ligand-dependent effect affecting the P2Y 6 receptor internalization and endosomal trafficking. These findings could guide the development of bias ligands that could influence P2Y 6 signaling. 相似文献
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